ABSTRACT Self-renewing and multipotent intestinal stem cells (ISCs) support homeostatic and injury-induced regeneration of the intestinal epithelium. The mechanisms that regulate the functions of ISCs under such conditions remain poorly understood and represent a topic of great interest. Recently, increasing evidence supports a model whereby ISCs exist in one of two functional states. These include an actively cycling ?aISC? state which drives normal renewal of the differentiated and post- mitotic intestinal lineages during homeostasis, and a quiescent reserve ?rISC? state which can be mobilized under injury conditions to re-enter the cell cycle and repair damaged epithelium. The process through which ISCs interconvert between rISC and aISC states is called ?state-switching?, is proposed to be reversible, and is likely fundamental to how intestinal stem cells balance self-renewal with differentiation and mount adaptive responses to tissue injury. Our goal is to understanding the regulatory mechanisms that regulate ?state-switching? in ISC. Preliminary data from the Magness lab and others indicates that genetic loss of Sox9 in intestinal epithelium causes ISC to adopt a highly proliferative aISC-like state that is incapable of regeneration after damaging insults such as radiation. Further, our preliminary data indicate that the rISC state is associated with high levels of Sox9 expression, whereas aISC express low levels of Sox9. The central hypothesis of this proposal is that Sox9 is an intrinsic master regulator of ISC state-switching, and directs aISC-rISC interconversions through high and low Sox9-expression thresholds. To test this hypothesis, I will use in vivo genetic models and in vitro high throughput single-cell culture methods to assess the consequences of experimentally-controlled Sox9 levels on ISC proliferation, intestinal lineage allocation, and resistance to radiation, the latter of which is a defining property of ?reserve? stemness. The studies proposed here will test if and how Sox9 expression-thresholds diversify stem cell functions in ISCs.